Src family kinase inhibitor PP1 improves motor function by reducing edema after spinal cord contusion in rats.

Following spinal cord injury vascular permeability increases around the area of injury, which possibly leads to secondary tissue damage. Vascular endothelial growth factor (VEGF) and Src which exists downstream of VEGF may contribute to edema formation. We here report that the Src family kinase inhibitor PPI could reduce edema and the inflammatory response after spinal cord injury. In this study we have examined the effect of PPI on motor function after mild spinal cord compression injury. We utilized a mild spinal cord compression model in rats. PPI or vehicle only was administered intraperitoneally after cord compression. The motor function of the hind limbs after injury was categorized into 7 grades. At 1, 3, 7 and 14 days after injury, the spinal cord was removed and the extent of edema formation and inflammation were examined using immunohistochemistry with an anti-IgG and anti-ED-1 antibody. The immunohistochemical analysis revealed that the area of edema formation and inflammation was remarkably reduced in animals with PPI. The muscle function was flaccid in both groups immediately after injury. However, at 3 and 8 days after injury, a significant improvement was observed in the PPI group. These results suggest that PPI is a strong candidate for drug treatment of spinal cord injury.

PTFE-fascia patch inlay method for the anterior approach for cervical intradural spinal lesion.

STUDY DESIGN: A new method for prevention of cerebrospinal fluid leakage was studied. OBJECTIVE: To prevent cerebrospinal fluid leakage, we developed a polytetrafluoroethylene fascia patch inlay method. BACKGROUND: One of the major risks of the anterior approach for intra-dural spinal cord lesions is the cerebrospinal fluid leakage. METHODS: A small hemangioblastoma located on the ventral side of the cervical cord was resected with an anterior approach. The dural closure was performed using this polytetrafluoroethylene fascia patch inlay method. RESULTS: The patient had a satisfactory clinical course with no cerebrospinal fluid leakage. CONCLUSION: This polytetrafluoroethylene fascia patch inlay method is very effective. This method may provide both prevention of CSF leakage and future spinal cord adhesion to the dura.

Effect of subconjunctival steroid injection on intraocular inflammation and blood glucose level after cataract surgery in diabetic patients.

PURPOSE: To prospectively evaluate the usefulness of a subconjunctival steroid injection given at the completion of cataract surgery in patients with diabetes mellitus. SETTING: University of Tokyo School of Medicine, Tokyo, Kaiya Eye Clinic, Hamamatsu, and Jyosai Hospital, Tokyo, Japan. METHODS: One hundred four eyes of 104 diabetic patients having routine small incision cataract surgery were randomized into 2 groups. One group received a subconjunctival injection of dexamethasone and the other group did not. Aqueous flare intensity was measured with the laser flare meter preoperatively and 1, 2, 5, 7, and 14 days postoperatively. Another 19 diabetic patients having routine cataract surgery were randomized to receive a subconjunctival steroid injection or not; blood glucose concentration was measured 4 times a day for 3 days postoperatively. RESULTS: There was no significant difference between the 2 groups in aqueous flare values at any postoperative time. The subconjunctival steroid injection induced a transient but significant increase in blood glucose on the day of surgery. CONCLUSION: A subconjunctival steroid injection given at the completion of cataract surgery in diabetic patients had no beneficial effects.

Anterior capsular contraction after cataract surgery in vitrectomized eyes.

PURPOSE: To assess the contraction of continuous curvilinear capsulorhexis after cataract surgery in eyes with past pars plana vitrectomy. METHODS: In a prospective study, 16 eyes of 16 patients underwent phacoemulsification and implantation of a foldable acrylic intraocular lens after pars plana vitrectomy. Eyes after intensive or repeated vitrectomy were not included. Twenty eyes of 19 patients served as age-matched controls. Aqueous flare intensity was measured using the laser flare-cell meter 1 year after surgery. The area of anterior capsular opening (ACO) was determined by diaphanoscopy using the anterior eye segment analysis system EAS-1000 at 1 day and 1 year postoperatively. RESULTS: There was no significant difference in the mean ACO area between the vitrectomy and control groups both at 1 day and 1 year postoperatively. Aqueous flare intensity 1 year after surgery was slightly higher in the vitrectomy group, but the difference was not statistically significant. CONCLUSION: Eyes after simple vitrectomy are not at a higher risk of ACO contraction following cataract surgery.

Anterior capsular contraction after cataract surgery in eyes of diabetic patients.

AIM: To investigate change in the area of anterior capsular opening (ACO) after cataract surgery and its relation to the degree of postoperative anterior inflammation in patients with diabetes mellitus (DM). METHODS: 31 eyes of 31 patients with DM and 30 eyes of 30 normal controls scheduled to undergo cataract surgery were examined prospectively. The area of ACO was measured with an anterior eye segment analysis system (EAS-1000) on the day following surgery and 3, 6, and 12 months after surgery. Comparative analyses were made on the area of ACO relative to the presence of DM and diabetic retinopathy (DR). The percentage reduction of area of ACO was calculated from values 1 day and 12 months after surgery, and multiple regression analysis was performed on the presence of DM, patient age, ACO area on the first postoperative day, and aqueous flare intensity 1 day and 12 months after surgery. RESULTS: The area was significantly smaller in the DM group at 3 (p=0.015, Student's t test), 6 (p=0.011), and 12 (p=0.010) months postoperatively. Patients having DR showed significantly smaller ACO area than the non-DR group 3 (p=0.039), 6 (p=0.033), and 12 (p=0.028) months after surgery. Multiple regression analysis revealed that presence of DM (p=0.003) and aqueous flare intensity 12 months after surgery (p=0.039) significantly correlated with the percentage reduction of area of ACO. Age, ACO area at 1 day postoperatively, and aqueous flare intensity immediately after surgery were not relevant to ACO contraction. CONCLUSIONS: Anterior capsular contraction after cataract surgery was greater in eyes of DM patients, especially in those with DR and increased permeability of the blood-aqueous barrier.

Synergistic antiproliferative effect of delta 12-prostaglandin J2 (delta 12-PGJ2) and hyperthermia on human esophageal cancer cell lines.

delta 12-PGJ2, one of the cyclopentenone prostaglandins and the ultimate metabolite of prostaglandin D2, has been reported to have potent antiproliferative activity on various tumor cells in vitro and in vivo. In this study, the combined effect of delta 12-PGJ2 and hyperthermia on six established cell lines of human esophageal carcinoma (SGF series) was analyzed by an in vitro assay, and the degree of apoptosis induced by this combination was examined to clarify the mechanism of supra-additive effects. In five SGF cell lines, except SGF-7 cells, combination therapy with delta 12-PGJ2 and hyperthermia showed synergistic antiproliferative effects. The supra-additive combined effect of delta 12-PGJ2 and hyperthermia on esophageal cancer cells is attributed to the synergistic induction of apoptosis. delta 12-PGJ2 induced G1 accumulation and apoptosis was induced by delta 12-PGJ2 from G1 phase. Hyperthermia induced G1 accumulation and apoptosis was induced by hyperthermia during all cell phases. Both augmented G1 arrest followed by G1 phase-selective induction of apoptosis and increased apoptotic induction without cell-cycle specificity are responsible for the synergism of combined treatment with delta 12-PGJ2 and hyperthermia.

Changes in tissue-plasminogen activator mRNA expression following cortical ablation in the rat brain.

Tissue plasminogen activator (tPA) has been used to treat acute thrombotic lesions. Roles other than the activation of fibrinolytic pathways have been suggested for tPA in the mature brain. We used the in situ hybridization technique to investigate the changes in tPA mRNA expression within the brain after cortical ablation. We found that expression of tPA mRNA started to increase diffusely in the cortex ipsilateral to the injury 6 h after ablation. This increase had become prominent 24 h after ablation. On d 5, the expression of tPA mRNA had returned to that of the control animals except for the area near the injury. We also found that administration of MK-801 before injury suppressed the increase of tPA mRNA in the ipsilateral cortex. These results suggest that the increase in tPA mRNA is likely to be mediated via activation of NMDA receptors.

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration.

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Pupillary functions after cataract surgery using flexible iris retractor in patients with small pupil.

PURPOSE: To quantitatively assess pupillary functions after small pupil cataract surgery using the flexible iris retractor. METHODS: Subjects were 11 patients (12 eyes) with small pupils who underwent phacoemulsification and intraocular lens implantation. Pupils were enlarged using the flexible iris retractor intraoperatively, and postoperative iriscorder data were compared with the data of 20 normal controls who underwent standard phacoemulsification and intraocular lens implantation. RESULTS: Although pupillary area before light stimulus did not differ between the groups, contraction rate after light stimulus was significantly lower in the small pupil group than in the normal controls. The velocity of contraction and dilation was also significantly slower in the small pupil group. Wider pupillary stretching during surgery resulted in deteriorated pupillary functions after surgery. Eyes of patients on long-term miotic therapy with pilocarpine showed poorer pupillary reaction postoperatively. CONCLUSION: Inappropriate use of the flexible iris retractor causes an atonic, chronically enlarged postoperative pupil. To avoid postoperative pupillary complications, miotic pupils should not be stretched to larger than a 5.0 x 5.0 mm square.

Expression of growth inhibitory factor mRNA after focal ischemia in rat brain.

Growth inhibitory factor (GIF) is a small protein belonging to the metallothionein family that has the capacity to inhibit neuronal survival and neurite formation in vitro. This study was conducted to investigate the role of GIF in the brain afflicted with ischemic injury. We used the in situ hybridization technique and Northern blot analysis to study the changes in GIF messenger RNA (mRNA) expression in a rat focal ischemia model. On the first day, the expression tended to decrease in the hemisphere ipsilateral to the injury. It returned to normal levels on the second day except for the central area of the middle cerebral artery (MCA) territory. On the third and fourth day, the expression increased diffusely in the hemisphere of the affected side, including the subcortical area. Two weeks after ischemia, the GIF mRNA expression increased again but only in the peri-infarcted area. Down-regulation of GIF on the first day in the cortex ipsilateral to the infarction might promote neurite sprouting. The subsequent increase in GIF mRNA expression on the third and fourth day might be a symptom of neurons attempting to inhibit excessive neurite outgrowth, or to protect themselves against toxicity caused by oxygen radicals. The later increase in the limited area around the infarction may be related to astroglial reaction. Growth inhibitory factor may play an important role in regulating the central nervous system after ischemic insults.

Loss and apoptosis of smooth muscle cells in intracranial aneurysms. Studies with in situ DNA end labeling and antibody against single-stranded DNA.

Pathological specimens were collected from 14 unruptured and 13 ruptured aneurysms at the time of clipping and studied in order to assess the underlying mechanism of rupture by investigating degeneration of the aneurysmal wall and possible involvement of apoptosis. Immunohistochemistry with anti-actin antibody showed few smooth muscle cells in the ruptured aneurysms and replacement of the muscularis layer by a fibro-hyalin tissue. However, at least one layer of smooth muscle cells was clearly observed in the unruptured aneurysms. Thus, smooth muscle cells in the wall of the ruptured aneurysms were much more degenerated than those in the wall of unruptured aneurysms. In addition, unruptured aneurysms with an angiographically smooth wall showed well-layered positive staining for anti-smooth muscle actin antibody while those with irregular shapes rarely reacted. We found, for the first time, evidence of DNA fragmentation in the aneurysmal wall. Apoptotic bodies were detected by means of a terminal transferase (TdT)-mediated dUTP biotin nick end labelling technique (TUNEL) and an anti-single-stranded DNA antibody in 54% (7/13) of the ruptured aneurysms. In contrast, apoptotic bodies were found in only 7% (1/14) of the unruptured cases. These results suggest that apoptotic cell death might be involved in the rupture of aneurysms.

Expression of tPA mRNA in the facial nucleus following facial nerve transection in the rat.

Plasminogen activators (PAs) have been suggested to play a role in neuronal migration and glial cell proliferation in the developing CNS. Less is known, however, about the role of PAs in the mature nervous system. To elucidate the role of tissue type plasminogen activator (tPA) in the nervous system we used in situ hybridization to study the expression of tPA mRNA within the rat facial nucleus after facial nerve transection. We also studied the effect of MK-801 on tPA mRNA expression in order to investigate whether the previously reported N-methyl-D-aspartate (NMDA) receptor activation is involved in this model. tPA mRNA was expressed in the ipsilateral facial motoneurones from 6 h after injury. This expression continued for at least 2 weeks after facial nerve transection. Administration of MK-801 before axonal injury did not affect the expression of tPA mRNA in the facial nucleus. These data suggest that tPA might be involved in the regenerative process without NMDA receptor activation in mature facial neurones.

Altered expression of Growth Inhibitory Factor (GIF / MT-III) mRNA in the rat facial nucleus after facial nerve injury is closely related with facial function.

Growth inhibitory factor / metallothionein III (GIF / MT-III) is reported to have the unique property of suppressing neuronal survival and neurite promotion in vitro. We investigated changes in the expression of GIF mRNA within the facial nucleus using in situ hybridization as well as changes in the function of the facial nerve after nerve injury. Following crushing injury just distal to the stylomastoid foramen, movement of the ipsilateral whiskers was eliminated but returned by the 7th day. GIF mRNA expression decreased at 3 days after injury and returned in 7 days. However, when the nerve was cut and sutured immediately, it took one month for the facial function to recover. In this case, GIF mRNA expression decreased 3 days after injury, remained at a low level for 14 days, and finally returned in 3-4 weeks. Thus, changes in the expression of GIF mRNA were found to be closely related to the facial nerve function.

Induction of Na+/myo-inositol cotransporter mRNA after focal cerebral ischemia: evidence for extensive osmotic stress in remote areas.

Myo-inositol is one of the major organic osmolytes in the brain. It is accumulated into cells through an Na+/ myo-inositol cotransporter (SMIT) that is regulated by extracellular tonicity. To investigate the role of SMIT in the brain after cerebral ischemia, we examined expression of SMIT mRNA in the rat brain after middle cerebral artery occlusion, which would reflect alteration of extracellular tonicity. The expression of SMIT mRNA was markedly increased 12 h after surgery in the cortex of the affected side and lasted until the second day. Increased expression was also found in the contralateral cingulate cortex. Up-regulated expression was found predominantly in the neurons in remote areas, although nonneuronal cells adjacent to the ischemic core also expressed this mRNA. These results suggest that cerebral ischemia causes extensive osmotic stress in brain and that the neuronal cells respond to this stress by increasing SMIT expression.

Changes in glutamate/aspartate transporter (GLAST/GluT-1) mRNA expression following facial nerve transection.

Expression of glutamate/aspartate transporter (GLAST/GluT-1) was investigated in the axotomized facial nucleus by in-situ hybridization. Hybridization signals for GLAST mRNA were almost undetectable in the facial nucleus of sham-operated animals. However, the hybridization signals were seen from 3 days after facial nerve transection onward in the nucleus of the affected side. These signals lasted at least 5 weeks. Microautoradiograms showed that small non-neuronal cells in the ipsilateral facial nucleus expressed signals of GLAST mRNA after axotomy. These findings suggest that non-neuronal cells, presumably astrocytes, may protect axotomized motor neurons against glutamate toxicity via up-regulation of GLAST in the facial nucleus.

Brief exposure to hypoxia induces bFGF mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress.

We examined the hypoxic tolerance phenomenon in vitro. Brief exposure to hypoxia induced the production of basic fibroblast growth factor (bFGF) mRNA and protein in rat cortical neurons and protected them from hypoxic injury. Cortical neurons were cultured from 18th-day rat embryos in a serum-free medium and subjected to brief (4 h) and/or prolonged (24 h) hypoxia. Neuronal damage was assessed by quantifying lactate dehydrogenase (LDH) activity in the medium. After brief hypoxia, LDH release was identical to that of the controls, whereas prolonged hypoxia caused a significant increase in LDH release, indicating neuronal death. However, if brief hypoxia was applied 2 days prior to the prolonged hypoxia, no increase in LDH release was observed. The bFGF mRNA expression was assessed with Northern blot and protein immunoreactivity with Western blot analysis. The brief period of hypoxia caused a 2.5-fold increase in bFGF mRNA and considerable bFGF protein expression 1 day later, but prolonged hypoxia caused increase in the expression of bFGF mRNA at 2 days and no protein expression until 3 days after the start of the hypoxia. When cells were subjected to prolonged hypoxia 2 days after brief hypoxia, however, no increase in bFGF mRNA was observed, while bFGF protein was expressed continuously. We also observed that exogenously applied bFGF reduced neuronal injury produced by prolonged hypoxia. The results obtained with this model suggest that brief hypoxia induces bFGF protein and thus tolerance to subsequent lethal hypoxia. Basic FGF might play a role as a tolerance-associated factor in this process. Thus, an in vitro model is useful for assessing the response of cortical neurons to hypoxic stress and for researching new factors related to ischemic tolerance.

Expression of growth inhibitory factor (GIF) in normal and injured rat brains.

Immunohistochemical study on growth inhibitory factor (GIF) in rat brain has revealed that a glial cell layer on the surface of cerebral cortex and the cells surrounding Purkinje cells has been reported. In addition, neurons in gray matter were weakly immunostained for GIF. In situ hybridization using digoxigenin-labeled single-strand RNA probes also demonstrated that most of the neurons and small round cells, which were presumably astrocytes, expressed GIF mRNA in the cerebral cortex of rat brain. These findings indicate that GIF is produced in neurons as well as in astrocytes. The most prominent findings in this study are, a very strong reaction of GIF and GIF mRNA in the reactive astrocytes around the site of injury induced by stab wound or kainic acid injection. These results raised the possibility that GIF may act as an acute-phase protein in reactive astrocytes and have a role in tissue repair.

Expression of growth inhibitory factor mRNA following cortical injury in rat.

Growth inhibitory factor (GIF) inhibits survival and neurite formation of cortical neurons in vitro and is found abundantly in the normal human brain. The role of GIF is still obscure, although it is reported to decrease in the brain in Alzheimer's disease. We examined changes in GIF mRNA expression in a rat cortical-ablation model with the aid of an in situ hybridization technique. In sham-operated animals, the GIF mRNA was expressed consistently in the cerebral cortex, hippocampus, and thalamus. One day after cortical ablation of the left somatosensory cortex, the expression tended to decrease in the cortex ipsilateral to the injury. Four days after surgery, it increased markedly in the affected cortex and thereafter returned to the level of the control animals except for the area surrounding the injury, where GIF mRNA again increased 2 to 3 weeks after ablation. The transient increase in GIF mRNA expression may reflect efforts to inhibit excessive sprouting of neurites. We also studied the effect of topically applied basic fibroblast growth factor (bFGF), which has a range of neurotrophic effects, on GIF mRNA expression. Topically applied bFGF enhanced the suppression of GIF at 1 day after surgery, though it did not affect the subsequent response. GIF can therefore be assumed to affect the outgrowth of injured neurites and might play a major role in maintenance of the neuronal network in cooperation with other trophic factors. Modification of these factors may be the key to improve neuronal damage after injury.

Changes in growth inhibitory factor mRNA expression compared with those in c-jun mRNA expression following facial nerve transection.

We investigated growth inhibitory factor (GIF) mRNA expression within the rat facial nucleus with the aid of in situ hybridization. We found that GIF mRNA was expressed abundantly in the facial motoneurons of sham operated animals, and that this gene expression decreased after transection of the facial nerve. This decrease of GIF mRNA was first detected on the third day and was maintained for at least five weeks after transection of the nerve. Changes in c-jun, an immediate early gene, were also investigated with this model, and it was found that c-jun mRNA started to increase in the facial nucleus on the first day and that this increase was maintained for at least 5 weeks. These results suggest that the facial motoneurons, when their axons are transected, continuously respond to the injury and that GIF mRNA is actively suppressed to reduce the inhibition of neurite outgrowth in order to regenerate the axons.

Expression of c-fos mRNA after cortical ablation in rat brain is modulated by basic fibroblast growth factor (bFGF) and the NMDA receptor is involved in c-fos expression.

Expression of c-fos mRNA after cortical injury was studied using the in situ hybridization technique. Strong signals for c-fos mRNA were observed immediately after cortical ablation in neurons throughout the cortex ipsilateral to the injury. However, this c-fos mRNA expression was transient and disappeared within 6 h after the injury. When basic fibroblast growth factor (bFGF; 1 micrograms) was applied to the site of ablation, c-fos mRNA signals were observed for a much longer period. Even 24 h after injury, diffuse expression of c-fos mRNA was detected throughout the cortex, being mainly confined to non-neuronal cells. Intraperitoneal injection of MK-801 (3 mg/kg), a non-competitive NMDA receptor antagonist, suppressed the expression of c-fos mRNA after cortical ablation. It suppressed both the immediate and late expression induced by cortical ablation and bFGF. The immediate expression of c-fos in neurons is likely to be due to spreading depression, while neuronal-glial interactions would be involved in the mechanism of late c-fos expression by non-neuronal cells. Our results suggest that induction of c-fos after cortical injury can be modulated by topically applied bFGF and that the N-methyl-D-aspartate (NMDA) receptor is involved in c-fos expression not only caused by injury itself but also induced by injury and bFGF. As the immediate early genes regulate secondary gene responses, the induction of c-fos may contribute to neuronal plasticity and bFGF may enhance its effect.